Monoclonal antibodies can rapidly be produced by the mouse immune system for biological studies. In a clinical setting, however, the use of these murine antibodies can result in a human anti-mouse antibody response (HAMA). Chimeric antibodies can reduce anti-IgG responses in human, but murine v-domains may still have provocative T-cell epitope content, necessitating “humanization” of their framework regions.
Classical humanization generally begins by transferring all six murine complementarity determining regions (CDRs) onto a human antibody framework (Jones et al., Nature 321, 522-525 (1986)). These CDR-grafted antibodies generally do not retain their original affinity for antigen binding, and in fact, affinity is often severely impaired. Besides the CDRs, certain non-human framework residues must also be incorporated into the variable domains to maintain proper CDR conformation (Chothia et al., Nature 342:877 (1989)). The incorporation of murine residues at key positions in the human frameworks to restore function is generally referred to as “back-mutations.” Back-mutations can support structural conformation of the grafted CDRs and restore antigen binding and affinity. Many of the framework positions that are likely to affect affinity have been identified, thus structural modeling to select new residues in a stepwise fashion can generally lead to variants with restored antigen binding. Alternatively, phage antibody libraries targeted at these residues can also be used to enhance and speed up the affinity maturation process (Wu et al., J. Mol. Biol. 294:151-162 (1999) and Wu, H., Methods in Mol. Biol. 207:197-212 (2003)).
Current humanization techniques still suffer from flaws, such as high non-human amino acid content retention; grafting into poorly understood frameworks; requirement for homology modeling of the v-domains, which is often inaccurate; or a co-crystal structure with the target antigen. Therefore, there is a need to develop new humanization methods.